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Sensitive fluorescence detection of polyphosphate in polyacrylamide gels using 4′,6‐diamidino‐2‐phenylindol
Author(s) -
Smith Stephanie A.,
Morrissey James H.
Publication year - 2007
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200700041
Subject(s) - dapi , polyphosphate , staining , nucleic acid , polyacrylamide , chemistry , fluorescence , glycosaminoglycan , biochemistry , metachromasia , toluidine , chromatography , microbiology and biotechnology , phosphate , biology , organic chemistry , polymer chemistry , physics , quantum mechanics , genetics , apoptosis
Abstract PAGE is commonly used to identify and resolve inorganic polyphosphates (polyP). We now report highly sensitive and specific staining methods for polyP in polyacrylamide gels based on the fluorescent dye, 4′,6‐diamidino‐2‐phenylindol (DAPI). DAPI bound to polyP in gels fluoresced yellow while DAPI bound to nucleic acids or glycosaminoglycans fluoresced blue. Inclusion of EDTA prevented staining of glycosaminoglycans by DAPI. We also identified conditions under which DAPI that was bound to polyP (but not nucleic acids or other anionic polymers) rapidly photobleached. This allowed us to develop an even more sensitive and specific negative staining method that distinguishes polyP from nucleic acids and glycosaminoglycans. The lower LOD using DAPI negative staining was 4 pmol (0.3 ng) phosphate per band, compared to conventional toluidine blue staining with a lower LOD of 250 pmol per band.